RESUMO
The binding of labeled insulin to dissociated R3230AC mammary adenocarcinoma cells from diabetic and intact rats was investigated in vitro. At 20 degrees, specific binding (total binding minus binding in the presence of 1000-fold excess or 10(-6) M unlabeled insulin) reached a plateau at 45 to 60 min and was directly related to the number of cells used. Degradation of labeled insulin, as measured by trichloroacetic acid precipitation, was related to the number of cells used, was not prevented by trasylol or phenylmethylsulfonyl fluoride (general proteolytic enzyme inhibitors), but was prevented by addition of 1 to 2% bovine serum albumin to the incubation medium. Specificity of insulisulin, and desoctapeptide insulin were capable of competing for insulin binding in an order of potency related to their relative biological activity; prolactin and glucagon were unable to compete for insulin binding. Scatchard analysis of the binding data demonstrated a curvilinear-plot; specific binding (over the concentration range of 10(-11) to 10(-10) M insulin) showed a high affinity (Kd approximately 1 to 3 X 10(-10) M), and the estimated number of sites was greater in tumors from diabetic animals than in tumors from intact animals or intact animals given insulin prior to sacrifice. Reversibility of insulin binding was studied by dissociation experiments; dissociation was enhanced in the presence of added unlabeled insulin compared to dissociation examined under conditions of "infinite" dilutions only. Maximum dissociation of bound insulin was observed in the presence of 10(-7) M unlabeled insulin, with less of an effect at lower or higher concentrations of added insulin (no effect seen at 10(-10) M insulin). Two techniques were investigated for separating cells from unbound labeled insulin; the procedure using centrifugation was found to be more efficient. Thus, in the R3230AC mammary adenocarcinoma, data obtained on saturability, reversibility, and specificity of insulin binding indicate the existence of an insulin receptor with properties similar to those found in normal cells.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Receptor de Insulina/metabolismo , Animais , Aprotinina/farmacologia , Sítios de Ligação , Ligação Competitiva , Contagem de Células , Centrifugação , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/metabolismo , Feminino , Filtração , Insulina/metabolismo , Insulina/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Ratos , Soroalbumina Bovina/farmacologia , Estreptozocina , Fatores de TempoRESUMO
Dissociated cells of the R3230AC mammary tumor were found to take up glucose by diffusion and by a passive carrier system. Using labeled 3-O-methylglucose as the probe, the following properties of the passive carrier were identified: (1) specificity for glucose, (2) competition by galactose and mannose but not by mannitol and fructose, (3) inhibition by phloretin but not by phloridzin, (4) temperature sensitivity, and (5) a Km for transport of 3-4 mM. The effects of insulin in vitro on carrier-mediated glucose transport were investigated in tumor cells from diabetic rats. At 10-9 M insulin, a time-related decrease in v for transport was observed resulting in an increased calculated Km (2- to 3-fold increase after 60-90 min incubation with insulin); only slight effects on V were obtained. This unusual response in v to insulin was observed when glucose was present in the medium at 2 mM and 5 mM, but not at 20 mM glucose. The effect of insulin to decrease the v was dose-related, with the major effects seen between 10-10M and 10-8M. The apparent decrease in glucose entry in vitro may in part explain the ability of insulin to inhibit growth of this tumor in vivo.
Assuntos
Adenocarcinoma/metabolismo , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Animais , Transporte Biológico , Diabetes Mellitus/induzido quimicamente , Difusão , Cinética , Masculino , Metilglucosídeos/metabolismo , Floretina/farmacologia , Ratos , EstreptozocinaRESUMO
Computer-assisted data analysis of binding isotherms (LIGAND) has shown that human platelets have binding sites for alpha-thrombin of high (Kd 0.3 nM), moderate (Kd 10 nM), and low affinities (Kd 3 microM). Application of similar techniques has shown that TLCK-thrombin does not, whereas PPACK-thrombin does, bind to the high-affinity binding site accessible to alpha-thrombin, but that both bind to the moderate and low-affinity sites. Treatment of platelets with Serratia marcescens protease destroys the high-affinity site but does not affect moderate-affinity binding. In accordance with this model, both modified thrombins compete with alpha-thrombin for platelet activation at the moderate-affinity site, but only PPACK-thrombin competes at the high-affinity site. These results establish that platelet activation by either low or moderate concentrations of thrombin are receptor-mediated events and explain the paradox of the differential effects of TLCK-thrombin on the binding and activation of platelets by alpha-thrombin.
Assuntos
Plaquetas/fisiologia , Receptores de Superfície Celular/fisiologia , Trombina/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Membrana Celular/metabolismo , Humanos , Técnicas In Vitro , Peso Molecular , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Trombina , Serotonina/metabolismo , Trombina/antagonistas & inibidores , Tosilina Clorometil Cetona/farmacologiaRESUMO
The technique of radiation inactivation has been used on highly purified human placental insulin receptor in order to determine the functional molecular size responsible for the insulin binding and to evaluate the "affinity regulator" hypothesis, which has been proposed to explain the increase in specific insulin binding to rat liver membranes observed at low radiation doses [Harmon, J. T., Hedo, J. A., & Kahn, C. R. (1983) J. Biol. Chem. 258, 6875-6881]. Three different types of inactivation curves were observed: (1) biphasic with an enhanced binding activity after exposure to low radiation doses, (2) nonlinear with no change in binding activity after exposure to low radiation doses, and (3) linear with a loss in the binding activity with increasing radiation exposures. A monomer-dimer model was the simplest model that best described the three types of radiation inactivation curves observed. The model predicts that an increase in insulin binding activity would result after exposure to low radiation doses when the initial dimer/monomer ratio is equal to or greater than 1 and a monomer is more active than a dimer. The monomer size of the binding activity was estimated to be 227,000 daltons by this model. This value most likely reflects the size of the monomeric alpha beta form. To substantiate this model, the purified receptor was fractionated by Sepharose CL-6B chromatography. The insulin binding profile of this column indicated two peaks.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Modelos Teóricos , Placenta/metabolismo , Receptor de Insulina/efeitos da radiação , Membrana Celular/metabolismo , Relação Dose-Resposta à Radiação , Feminino , Humanos , Insulina/metabolismo , Cinética , Substâncias Macromoleculares , Gravidez , Receptor de Insulina/isolamento & purificação , Receptor de Insulina/metabolismoRESUMO
Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation.
Assuntos
Clorometilcetonas de Aminoácidos/metabolismo , Plaquetas/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Trombina/análogos & derivados , Trombina/farmacologia , Tosilina Clorometil Cetona/metabolismo , Sítios de Ligação , Ligação Competitiva , Plaquetas/metabolismo , Humanos , Técnicas In Vitro , Ligantes , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Trombina , Serotonina/metabolismo , Trombina/metabolismo , Tosilina Clorometil Cetona/análogos & derivadosRESUMO
The functional sizes of the binding sites for thrombin on human platelets and isolated membranes have been determined by the technique of radiation inactivation: similar results were obtained. Independent studies using different radiation doses (0, 3, and 48 Mrad) and different thrombin concentrations (10(-10), 10(-8), and 10(-6) M) confirmed the presence of three binding sites with functional sizes of 900 000, 30 000, and 4000 daltons. The binding site of lowest apparent size (4000 daltons) probably corresponds to what has been termed nonspecific binding since its dissociation constant (2900 nM) is well outside the physiological range. The site of intermediate size (30 000 daltons) is also probably not involved in platelet activation since its dissociation constant (11 nM) is also beyond the concentration range required for activation, although it may be involved in other aspects of platelet-thrombin interaction. The sites with the largest functional size are probably important in platelet function since their dissociation constant (0.3 nM) is in the range required for platelet activation. The functional size of these sites (900 000 daltons) suggests that the high-affinity site for thrombin binding to platelets may involve a multimolecular complex of membrane components.
Assuntos
Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Trombina/metabolismo , Membrana Celular/metabolismo , Humanos , Radioisótopos do Iodo , Cinética , Peso Molecular , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/efeitos da radiação , Receptores de TrombinaRESUMO
A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.
Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombina/metabolismo , Animais , Anticorpos , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Proteínas de Membrana/metabolismo , Octoxinol , Polietilenoglicóis , Coelhos , Ensaio Radioligante , SolubilidadeRESUMO
The receptor status of the moderate-affinity platelet binding site for alpha-thrombin has been established by treating platelets with Serratia marcescens protease under conditions causing cleavage of 95-97% glycoprotein Ib (2.5 micrograms for 30 min). High-affinity binding was lost under these conditions, but the platelets continued to show moderate-affinity binding (Kd1 = 16 +/- 5 nM; 930 +/- 300 sites/platelet) and low-affinity binding (Kd2 = 4.6 +/- 3 microM; 170,000 +/- 90,000 sites/platelet), in good agreement with the values previously obtained for moderate- and low-affinity binding in intact platelets [Harmon, J.T., & Jamieson, G.A. (1986) J. Biol. Chem. 261, 15928-15933]. Platelets treated with Serratia protease under these conditions were about 4-fold less sensitive to activation by alpha-thrombin, as measured by serotonin secretion. Crossover studies with analogues showed that binding of alpha-thrombin was compatible by both D-phenyl-alanyl-L-prolyl-L-arginine chloromethyl ketone treated thrombin and N alpha-p-tosyl-L-lysine chloromethyl ketone treated thrombin, and both analogues were capable of inhibiting activation of Serratia-proteolyzed platelets by alpha-thrombin. These studies establish that the moderate-affinity platelet binding site for alpha-thrombin is a receptor, occupancy of which is required for platelet activation in the absence of the high-affinity receptor.
Assuntos
Plaquetas/fisiologia , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores de Superfície Celular/fisiologia , Trombina/fisiologia , Humanos , Cinética , Modelos Biológicos , Peptídeo Hidrolases , Receptores de Trombina , Serotonina/sangue , Serratia marcescens/enzimologiaRESUMO
Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The Kd for insulin binding, approximately 10(--10) M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3-0-methyl glucose (3-OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 X 10(--9) M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in Km and Vmax. Insulin decreased initial velocity of glucose transport at 4--6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed.
Assuntos
Adenocarcinoma/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Receptores de Superfície Celular , Animais , Sítios de Ligação , Transporte Biológico , Transporte Biológico Ativo , Glicemia/metabolismo , Difusão , Feminino , Insulina/sangue , Cinética , Metilglucosídeos/metabolismo , Proteínas de Neoplasias/metabolismo , Ligação Proteica , RatosRESUMO
Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3-aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2',3'-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5'-p-fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2',3'-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Receptores Purinérgicos/metabolismo , Difosfato de Adenosina/análogos & derivados , Ligação Competitiva , HumanosRESUMO
The technique of radiation inactivation has been used on a highly purified insulin receptor in order to determine the functional molecular size responsible for tyrosine-specific protein kinase activity. When both insulin binding and kinase activities were analyzed with the same receptor preparations, the functional size for kinase activity was found to be larger than that for insulin binding activity. The radiation inactivation curve for kinase activity was multiphasic. This indicates that at least two components contribute to total kinase activity. The average minimal functional size for the kinase was 370,000 +/- 60,000 daltons (n = 7) which corresponds to the alpha 2 beta 2 form of the insulin receptor. The average functional size for larger forms was estimated to be approximately 4 X 10(6) daltons. (To minimize the complexity of the model used in this analysis, we have analyzed the radiation inactivation curves of the insulin receptor kinase activity with a two-component model. However, we believe that the larger component, greater than 1 X 10(6) daltons, is probably not a single molecular weight species but rather represents a continuum of sizes or aggregates of the alpha 2 beta 2 form of the receptor.) These larger forms contributed 93% of the total activity. Mild reduction of the insulin receptor preparation with dithiothreitol (DTT) activated the total kinase activity by 3.5-fold. Under this condition, the minimal functional kinase size was 380,000 +/- 30,000 daltons (n = 6) while the average functional size for the larger forms was approximately 3 X 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas Tirosina Quinases/efeitos da radiação , Receptor de Insulina/efeitos da radiação , Animais , Sítios de Ligação , Cromatografia em Gel , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos da radiação , Insulina/análise , Matemática , Tamanho da Partícula , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Relação Estrutura-Atividade , Tirosina/farmacologiaRESUMO
Using the technique of radiation inactivation we have previously shown that the insulin receptor behaves as if it is composed of at least two functional components: a binding component (Mr approximately equal to 100,000) and an affinity regulatory component (Mr approximately equal to 300,000). The interaction between the affinity regulator and binding component results in a decrease in the affinity of the receptor for insulin. To examine in more detail the interaction between this "affinity regulator" and the binding component we have studied the insulin receptor by radiation inactivation under conditions which alter receptor concentration or receptor affinity. Liver membranes of ob/ob mice exhibit a decrease in insulin binding when compared to their lean litter mates which is due to a decrease in receptor concentration. When studied by radiation inactivation, however, there was no detectable change in the interaction or size of the two receptor components. By contrast, under circumstances in which the affinity of the receptor was increased (treatment with high salt, high pH, 1 mM dithiothreitol, 1-5 micrograms/ml of trypsin), the interaction between the regulatory and binding components was either decreased or absent, i.e. there was no increase in binding with irradiation. Conversely, conditions which produce a decrease in receptor affinity resulted in an increase in the interaction between the regulatory and binding components. The changes in receptor affinity and interactions of the two components produced by either high salt or pH were reversible. Partial purification of the solubilized receptor on lectin affinity columns resulted in the apparent removal of the affinity regulator, i.e. receptor affinity was increased. In this state, radiation inactivation studies revealed a monoexponential decay indicating no interaction between binding and regulatory components. Taken together, these results suggest that the affinity regulator is a membrane protein which is both trypsin-sensitive and has disulfide bond(s) essential for its function. The interaction between the affinity regulator and binding component is not via a covalent bond and the two components appear to be separated by lectin chromatography. The interaction between these components appears to be altered in most states associated with altered receptor affinity.
Assuntos
Fígado/metabolismo , Receptor de Insulina/metabolismo , Animais , Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Insulina/análogos & derivados , Insulina/metabolismo , Cinética , Substâncias Macromoleculares , Camundongos , Camundongos Obesos , Peso Molecular , Ratos , Ratos Endogâmicos , Tripsina/farmacologiaRESUMO
Activities of the various components of the human factor VIII complex in citrated and heparinized human plasma have been determined following radiation inactivation of the plasma in a high energy electron beam at -135 degrees C in order to determine the molecular size of the functional units. In citrated and in heparinized plasma the functional size of VIII:C was 120,000 +/- 9,700 and 140,000 +/- 10,000, respectively. Taken together with previously published data, these results suggest that VIII:C exists in plasma as a dimer of noncovalently bonded functional subunits. The size of the functional unit of the ristocetin cofactor of the factor VIII complex was determined as being approximately 330,000 in both citrated and heparinized samples. Immunological assays for VIII:C (inhibitor neutralization assay), the VIII:C antigen, and the VIII:vWF-related antigen suggest that these may not be reliable under conditions favoring the activation and inactivation of factor VIII components by thrombin or other proteases.
Assuntos
Fator VIII , Elétrons , Fator VIII/efeitos da radiação , Heparina , Humanos , Substâncias Macromoleculares , Peso MolecularRESUMO
Using the technique of radiation inactivation we have previously shown that the insulin receptor in rat liver membranes is composed of at least two functional components: an insulin binding component and a component which acts as an affinity regulator ((1980) J. Biol. Chem. 255, 3412-3419). In the present study, we have used this technique to examine whether insulin alters the size or structure of the insulin receptor. When insulin is bound to its receptor in liver membranes before radiation, there is a marked change in the inactivation profile. Analysis of the results of indicated that insulin binding to its receptor caused a decrease in the functional size of the insulin binding component from 115,000 to 76,000 and also diminished the interaction between the binding and regulatory components. The magnitude of these effects depended on the insulin concentration, and analogues of insulin with reduced receptor affinity show a reduced effect. Under these conditions, there was no change in the apparent size of the affinity regulator (about 260,000). These data suggest that the interaction of insulin with its receptor in situ produces a change in receptor structure which may be important in signal transmission.
Assuntos
Insulina/farmacologia , Fígado/metabolismo , Receptor de Insulina/efeitos dos fármacos , Animais , Membrana Celular/metabolismo , Insulina/análogos & derivados , Insulina/metabolismo , Cinética , Peso Molecular , Proinsulina/farmacologia , Ratos , Receptor de Insulina/metabolismo , Receptor de Insulina/efeitos da radiaçãoRESUMO
Platelets were obtained from patients with various hyperlipidemias [type II, type V, lecithin-cholesterol acyltransferase (LCAT) deficiency] and hypolipidemias (abetalipoproteinemia, Tangier disease) to ascertain relationships among plasma lipids, platelet lipids, thrombin binding and thrombin-induced platelet aggregation, and to compare these data with those previously obtained on stimulus-response coupling in platelets following in vitro modification of membrane microviscosity. Washed platelets were studied for their ability to bind 125I-thrombin in the range of 10(-10) to 10(-6) mol/L (10 mU/mL to 100 U/mL) and to aggregate with thrombin at concentrations less than 10(-9) mol/L (100 mU/mL). The values for binding and aggregation in eight patients from six kindred with familial hypercholesterolemia, taken as a group, fell in the low normal range. If divided into two groups, patients with overt cardiovascular disease bound normal amounts of thrombin but were more responsive to it, whereas patients without overt cardiovascular disease bound lower amounts of thrombin but gave an aggregation response in the normal range. These results suggest that platelet hyperresponsiveness in familial hypercholesterolemia arises from an alteration in the coupling mechanism between thrombin binding and response such that platelets from patients with familial hypercholesterolemia are able to respond with lower receptor occupancy than is the case with normal platelets. Thrombin binding and aggregation were within normal ranges for platelets from abetalipoproteinemia patients (N = 4) and type V hyperlipoproteinemia (N = 2), although in the latter case the response appeared to be less at very low thrombin concentrations (less than 30 mU/mL). Thrombin binding was elevated in Tangier disease (N = 3) but with lower responsiveness at lower thrombin concentrations. Thrombin binding was also elevated in LCAT deficiency (N = 2), and one patient showed increased and another showed decreased aggregation responses. In general, increased plasma cholesterol levels resulted in increased stimulus-response coupling (type II), whereas increased triglyceride levels resulted in decreased coupling (type V, Tangier), and there was no apparent alteration in the coupling mechanism with overall reduction in plasma lipid levels as in abetalipoproteinemia.
Assuntos
Plaquetas/metabolismo , Transtornos das Proteínas Sanguíneas/sangue , Lipoproteínas/sangue , Trombina/metabolismo , Abetalipoproteinemia/sangue , Adulto , Feminino , Humanos , Hiperlipoproteinemia Tipo II/fisiopatologia , Deficiência da Lecitina Colesterol Aciltransferase/fisiopatologia , MasculinoRESUMO
The microviscosity of human platelet membranes was changed by incubating platelets with liposomes containing various ratios of cholesterol and lecithin. Binding of 125I-thrombin to the modified platelets was measured together with platelet aggregation and secretion. In cholesterol-normal platelets (mole ratio of cholesterol to phospholipid (C:PL) = 0.553; eta = 2.40 poise), weighted nonlinear least squares curve fitting indicated that a model involving two classes of sites was adequate to describe the binding isotherm (K1 = 8.3 X 10(8) M-1; R1 = 150 sites/platelet; K2 = 6.4 X 10(6) M-1; R2 = 16,000 sites/platelet). In cholesterol-enriched platelets (C:PL = 0.857; eta = 3.05 poise), the apparent affinities for the two classes of sites decreased to 55 and 53%, respectively, while the binding capacities increased to 170 and 160%, respectively. In contrast, in the cholesterol-depleted platelets (C:PL = 0.435; eta = 2.03 poise), the affinities increased to 220 and 180%, respectively, while the binding capacities decreased to 53 and 46%, respectively. In cholesterol-enriched, cholesterol-normal, and cholesterol-depleted platelets, the thrombin concentrations required for half-maximal aggregation were 0.17, 0.35, and 0.52 nM, respectively, while the values for half-maximal secretion of [14C]serotonin were 0.17, 0.40, and 0.55 nM, respectively. Plots of receptor occupancy versus biological response showed that maximum response in cholesterol-enriched, cholesterol-normal, and cholesterol-depleted platelets occurred with occupancy of 30, 50, and 70% of the high affinity sites, respectively. In all three treatment groups, occupancy of 40-50 high affinity sites results in 50% aggregation. These results show that (i) modification of platelet membrane microviscosity results in changes in the number and affinity of both high and low affinity thrombin receptors, (ii) the change in receptor number rather than affinity is the determinant for platelet responsiveness, and (iii) the changes in membrane microviscosity do not appear to alter the coupling between occupied receptor and subsequent bioresponse.